An approach based on ultra-high pressure liquid chromatography–tandem mass spectrometry to quantify O6-methyl and O6-carboxymethylguanine DNA adducts in intestinal cell lines
Highlights
► Optimisation of the extraction of O6-carboxymethylguanine and O6-methylguanine. ► Development and proper validation of an UHPLC–MS/MS detection method. ► Working with pre-extracted DNA instead of cultured cell lines is more efficient. ► Applicability of the detection method for in vitro and in vivo experiments.
Introduction
Detection of DNA adducts is widely used for monitoring the exposure of cellular DNA to genotoxic agents. Knowledge of the nature and amounts of DNA adducts formed in vivo or in vitro provides valuable information regarding the mutational effects that may result from particular exposures. Epidemiological and clinical studies have consistently demonstrated that the consumption of meat and in particular red and processed meat is associated with an increased risk of colorectal cancer [1]. The formation of the DNA adducts O6-carboxymethyl-2′-deoxyguanosine (O6-CMdG) and O6-methyl-2′-deoxyguanosine (O6-MedG) has been associated with increased colorectal cancer risk upon intake of red and processed meat.
These particular DNA adducts (O6-CMdG and O6-MedG) are the result of endogenously formed alkylating N-nitroso compounds (NOCs), which are metabolised to highly reactive methylating agents that interact with the nucleophilic centres of DNA bases [2], [3]. In turn, the formation of these endogenous NOCs has been hypothesised as a plausible origin for increased colon cancer risk, since a dose–response relation with the faecal excretion of NOCs for red and processed meat but not for white meat intake has been established [4], [5], [6]. Evidence of human exposure to NOCs, reinforcing the presence of alkylating agents in the human gastrointestinal (GI) tract [7], [8] is based on the fact that O6-CMdG and O6-MedG are indeed detectable in colonic biopsies and human blood DNA samples [9], [10], [11].
The identification and quantification of very low DNA adduct concentrations, in vivo or in vitro requires ultrasensitive methodologies. This is particularly true for the analysis of human samples or in vitro applications on cell lines, where only small amounts of sample, and therefore DNA, are available. The methods currently used for DNA adduct determination include immunoassays [12], [13] and capillary electrophoresis-laser induced fluorescence immunoassays [14], 32P-postlabeling [15], GC/ECD [16] and HPLC with fluorescence detection [17], [18]. In recent years there has been a continuous improvement in the analytical approaches used to study these adducts, both qualitatively and quantitatively, with mass spectrometric detection playing an increasing role in these developments [19], [20], [21], [22]. HPLC coupled to mass spectrometric detection achieves a perfect balance between a high specificity and sensitivity. Specificity is further improved when utilising the selected reaction monitoring (SRM) mode of tandem triple quadrupole mass spectrometers. LC–MS has already been applied to both O6-MeG and O6-MedG adducts [21], [23], however to the best of our knowledge no studies so far have reported MS-based methods with combined detection of O6-methyl adducts and O6-carboxymethyl adducts either as the bases or the nucleosides.
For this reason, the aim of our present study was to investigate the potential of triple quadrupole mass spectrometry (QqQ-MS) coupled to ultra-high pressure liquid chromatography (UHPLC) to quantify in a highly sensitive and specific manner the DNA adducts of interest, O6-MeG and O6-CMG, in vitro in colonic cell lines treated with the known NOC potassium diazoacetate (KDA), and for future experiments in vivo in human exfoliated colonocytes. To this purpose, a sample pre-treatment and UHPLC–MS/MS detection method were developed followed by an extensive validation study performed with a standard reference DNA; calf thymus (CT) DNA. This newly developed UHPLC–MS/MS method was then applied in vitro to measure the generated adduct levels in NOC-treated human colon carcinoma cell lines (Caco-2 and HT-29).
Section snippets
Reagents and chemicals
Caution: KDA is carcinogenic. It should be handled in a well ventilated fume hood with extreme care and with personal protective equipment.
The chemical standard O6-MeG was purchased from Sigma–Aldrich (St. Louis, MO, USA) and the internal standard O6-methyl-d3-guanine (O6-Me-d3-G) was obtained from Toronto Research Chemicals Inc. (Toronto, Canada). O6-CMG was derived via 0.1 M formic acid hydrolysis at 70 °C for 1 h, from O6-CMdG with subsequent purification [12]. The stock solutions of the
Development of sample clean-up
To evaluate DNA adduct recovery, a proper execution of the DNA hydrolysis step is required. Mild acid hydrolysis of DNA samples with 0.1 M formic acid (F.A.) and hydrochloric acid (HCl) was compared for its purine base-releasing capacity [12]. Besides the type of acid used, a different hydrolysis time and temperature were investigated, respectively, 30 min at 80 °C and 60 min at 70 °C. The hydrolysed CT-DNA samples were analysed by UHPLC–MS/MS to investigate the O6-MeG and O6-CMG content. The rate
Discussion
The present study describes the optimisation and validation of a high-throughput, accurate and robust analysis method for the combined detection of O6-carboxy and O6-methylguanine DNA adducts. This quantitative analytical method has been subsequently applied to measure the DNA adduct formation in vitro on the human colonic carcinoma cell lines HT-29 and Caco-2 (and their pre-extracted DNA) after incubation with the known gastro-intestinal N-nitroso compound KDA and in vivo on human colonocytes
Conclusion
The results of the present study acknowledge the excellent applicability of triple quadrupole mass spectrometry coupled to ultra-high pressure liquid chromatography for the simultaneous detection of DNA adducts, more specifically O6-methylguanine and O6-carboxymethylguanine, in a accurate, sensitive, robust and high throughput manner. The performance characteristics (i.e. specificity, selectivity, repeatability, linearity, precision, recovery and limit of quantification) of this validated
Funding sources
L. Vanhaecke is a postdoctoral fellow from the Research Foundation – Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen).
Acknowledgment
The authors would like to thank M. Naessens, L. Dossche, J. Goedgebeur and D. Stockx for their practical assistance in the laboratory.
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